Cellular internalization of large hydrophilic therapeutic agents such as proteins or nucleic acids is still a challenging task because of the presence of plasma membrane, which constitutes an impermeable barrier for such molecules. In order to circumvent this problem, several methods of carrier-mediated delivery systems have been developed. Among them, much attention has recently been given to the use of peptide-based delivery systems. The use of peptides with cell-penetrating properties has several advantages, which are mainly due to various modifications that can be done to the peptide sequence. This allows the engineering of carriers addressing different cellular subdomains and/or is able to transport various types of cargos. Cell penetrating peptides (CPPs) such as antennapedia-derived penetratin (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J Biol Chem 269, 10444-50) and HIV Tat peptide (Vives, E., Brodin, P., and Lebleu, B. (1997) J Biol Chem 272, 16010-7) are widely used tools for the delivery of peptides, proteins and oligonucleotides into cells (Fischer, P. M., Krausz, E., and Lane, D. P. (2001) Bioconjug Chem 12, 825-41). Areas of application range from cell biology (Jarver, P., Langel, K., El-Andaloussi, S., and Langel, U. (2007) Biochem Soc Trans 35, 770-4) to biomedical research (Foerg, C., and Merkle, H. P. (2008) J Pharm Sci 97, 144-62). Immunological relevance of CPPs is crucial to biomedical research. Nevertheless, most CPPs used to date are of non-human origin and therefore an adaptive immune response may be induced, especially when conjugated to proteins and nanoparticles. Therefore, CPPs like the human calcitonin-derived peptide (Ding, G. J., Fischer, P. A., Boltz, R. C., Schmidt, J. A., Colaianne, J. J., Gough, A., Rubin, R. A., and Miller, D. K. (1998) J Biol Chem 273, 28897-905) and peptides corresponding to signal sequences of human proteins (Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y. Z. (1998) Nat Biotechnol 16, 370-5) are being considered as highly attractive import vehicles.
Human eosinophil cationic protein (ECP) secreted by activated eosinophil granules possesses 133 amino acids with an isoelectric point (pI) of 10.8 (Venge, P., Bystrom, J., Carlson, M., Hakansson, L., Karawacjzyk, M., Peterson, C., Seveus, L., and Trulson, A. (1999) Clin Exp Allergy 29, 1172-86) and a molecular weight ranging from 16 to 22 kDa as a result of different glycosylation extent (Venge, P., Bystrom, J., Carlson, M., Hakansson, L., Karawacjzyk, M., Peterson, C., Seveus, L., and Trulson, A. (1999) Clin Exp Allergy 29, 1172-86). ECP possesses antibacterial activity (Rosenberg, H. F. (1995) J Biol Chem 270, 7876-81) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus) and Gram-negative bacteria including Escherichia coli (E. coli) (Rosenberg, H. F. (1995) J Biol Chem 270, 7876-81). Besides bactericidal activity, ECP also has helminthotoxic (Hamann, K. J., Gleich, G J., Checkel, J. L., Loegering, D. A., McCall, J. W., and Barker, R. L. (1990) J Immunol 144, 3166-73) and antiviral (Domachowske, J. B., Dyer, K. D., Adams, A. G, Leto, T. L., and Rosenberg, H. F. (1998) Nucleic Acids Res 26, 3358-63) activities against respiratory syncytial virus (RSV). It has been proven that synthetic ECP(1-45) peptide reproduces most of ECP's antimicrobial properties and the ECP(24-45) segment is essential for antimicrobial activity (Torrent, M., de la Torre, B. G, Nogues, V. M., Andreu, D., and Boix, E. (2009) Biochem J 421, 425-34). We have previously reported that cell surface glycosaminoglycan (GAGs), especially heparan sulfate proteoglycans (HSPGs) promotes internalization of ECP via macropinocytosis pathway (Fan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y., and Chang, M. D. (2007) Traffic 8, 1778-95). The cytotoxicity of ECP was severely reduced toward the mutant cell lines deficient in heparan sulfate (HS) (Fan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y., and Chang, M. D. (2007) Traffic 8, 1778-95). Our results indicate that the motif containing charged and aromatic residues located within the region ECP(34-38) modulates the interaction between ECP and heparin (Fan, T. C., Fang, S. L., Hwang, C. S., Hsu, C. Y., Lu, X. A., Hung, S. C., Lin, S. C., and Chang, M. D. (2008) J Biol Chem 283, 25468-74). The motif has also been disclosed in the U.S. Pat. No. 7,595,374.
In addition, it has been demonstrated that antimicrobial peptides and CPPs possess concordant functional characteristics. For example, Magainin 2 from the skin of the South-African clawed frog Xenopus laevis is taken up into mammalian cells (Takeshima, K., Chikushi, A., Lee, K. K., Yonehara, S., and Matsuzaki, K. (2003) J Biol Chem 278, 1310-5) and shows antimicrobial activity (Nekhotiaeva, N., Elmquist, A., Rajarao, G K., Hallbrink, M., Langel, U., and Good, L. (2004) FASEB J 18, 394-6). In the present invention, the biological function and potential application of a novel CPP derived from ECP which contains a heparin binding motif are disclosed.